Diagnostic aids for use in the detection of bacteria in biological and other fluids

ABSTRACT

The invention is directed to novel compounds of the formula   WHERE X is oxygen, sulfur or an alkylated nitrogen atom and R is   WHERE R1 is an aliphatic, aromatic, araliphatic, acyl, hydroxy, or acyloxy aliphatic radical. R2 is hydrogen or lower alkyl, R3 and R4 are both hydrogen, or, together, an isopropylidene radical and n is 2 or 3; diagnostic agents containing said compounds for use in the detection of bacteria in biological and other fluids, and the process for detecting bacteria in such fluids using said agents which comprises incubating the fluid to be investigated in the presence of a nutrient medium and a compound as above set out, the number of bacteria being directly related to the color change thereafter observed.

United States Patent Herbert Berger Sulzberg-Rled Allgnug Otto Dold,Ilvesheim; Dietrich Kruger, Plankstadt/b, Schwetzingen; Kurt Stach,Mannheim; Felix Helmut Schmidt, Mannheim-Neuosthelm; Harald Stork,Lamperthelm, I-Iesen, all 01 Germany 812,501

Dec. 26, 1968 Nov. 16, 1971 Boehrlnger Mannheim Gesellschait mitbeschrankter IIaItung Mar. 29, 1966 Germany Sept. 8, 1966, Germany, No.B 88 815; Nov. 1 l, 1966, Germany, No. B 89 784 Original applicationFeb. 15, 1967, Ser. No. 616,201, now Patent No. 3,496,066, dated Feb.17, 1970. Divided and this application Dec. 26, 1968, Ser. No. 812,501

[72] Inventors Priorities DIAGNOSTIC AIDS FOR USE IN THE DETECTION Miuraet al., Yakugaku Zasshi, Vol. 84, pages 537 to 543 (1964) PrimaryExaminer.lohn D. Randolph Almrney-Burgess, Dinklage & Sprung ABSTRACT:The invention is directed to novel compounds of -t' ef rm9la,

oN- LR where X is oxygen, sulfur or an alkylated nitrogen atom and R,where R is an aliphatic, aromatic, araliphatic, acyl, hydroxy,

or acyloxy aliphatic radical. R, is hydrogen or lower alkyl, R; and Rare both hydrogen, or, together, an isopropylidene radical and n is 2 or3; diagnostic agents containing said compounds for use in the detectionof bacteria in biological and other fluids, and the process fordetecting bacteria in such fluids using said agents which comprisesincubating the fluid to be investigated in the presence of a nutrientmedium and a compound as above set out, the number of bacteria beingdirectly related to the color change thereafter observed.

DlAGNOSTlC AIDS FOh USE IN THE DETECTION OF BA l i iNBlOLOGlCALAND iFLUIDS This application is a division of Ser. No. 616,201 filed on Feb.15, 1967, now U.S. Pat. No. 3,496,066 issued Feb. I7, 1970.

The present invention relates to diagnostic agents for use in thedetection of bacteria, and to the methods for manufacturing and usingthe same. Still further, this invention relates to a class of novelcompounds which are metabolized by bacteria whereby metabolic productsconstituting dyestuffs are produced. The presence or absence of colorformation, as well as degree thereof, being directly related to theamount, if any, of bacteria present.

For the rapid and effective diagnosis and combating of infectiousdiseases, it is desirable to be able to detect as quickly as possibleboth qualitative and quantitatively the micro-organisms causing thedisease and further to be able to differentiate them with certainty. inaddition to the classical serological methods, a number ofinvestigational processes are known in which the enzymes formed by themicro-organisms are detected by means of a color indicator or in whichthe metabolic products of specific bacteriophages are utilized for thedetection of the bacteria.

Thus, for example, the detection of certain kinds of bacteria ispossible by means of the Griess nitrite test in which nitrate is reducedto nitrite by the nitrite reductasc formed by the bacteria, the presenceof nitrite being detected with sulfanilic acid and a-naphthylamine (HJ.Walther, Krztl. Lab., 6, 287/ 1960). Since this test constitutes only anonspecific and not very reliable means for the detection of nitrite anddoes not constitute a specific or direct detection of the bacteria, ithas only a limited and conditional importance (Linzenmeier et aL, Klin.Wschr., 41, 9l9/1963).

Another method which has been proposed for the detection of bacteriadepends upon the reduction of the colorless 2,3,5- triphenyl-tetrazoliumchloride to the red-brown colored triphenyl-formazane by reductasespeculiar to bacteria. This reaction is entirely nonspecific and,therefore, does not permit a differentiation of the bacteria present(Simmons et al., Lancet, l, l377/l962).

ln U.S. Pat. No. 3,122,480, there is disclosed a method for thedetection of strains of Pseudomonas which depends upon the reaction ofenzymes peculiar to bacteria with p-phenylenediamine and substitutednaphthol derivatives whereby colored compounds (indophenol blue) areproduced. In carrying out this detection method, the micro-organisms tobe identified must first be caused to grow by the use of selectivenutrient media, which requires incubation periods of about 18 hours at37 C. For the isolation of an important causative organism of infectionsof the urinary tract, namely, Pseudomonas acruginosn, very specialnutrient media are required, thereby rendering the use of this knownprocess even more difficult. However, the really important disadvantageof this process is the fact that Escherichia coli and the enterococci,such as Streptococcus faecalis, are not detected, although these twolatter organisms are found to be the causative organisms in more thantwo-thirds of all nonspecific infections of the urinary tract.

in U.S. Pat. No. 2,970,945 there has been described for use in thedetection of a special strain of bacteria, namely the gonococci,tetramethyl-p-phenylene-diamine, the indicator substance thereby beingoxidized to a dyestufl' by oxidases peculiar to the bacteria. Because ofthe atmospheric sensitivity of the indicator substance and of thelimitation to one strain of bacteria, a general importance cannot beattributed to this reaction.

Finally, a number of publications have appeared which describe processesfor the detection of bacteria wherein bacteriophages are utilized (l-Ll.Raettig, Bakteriophagie," pub. Gustav Fischer-Verlag, Stuttgart 1958,page 53). However, for several reasons, it has not been possible todevelop this method into one for routine use because "there are hardlyany monovalent bacteriophages for one strain of bacteria and these fewmonovalent phages easily become polyvalent and thus extend to otherbacterial strains." Furthermore, since bacteria are very frequentlyphage-resistant, the already very laborious and time-consuming detectionis, in addition, not dependable and is unsuitable in practice.

This invention has as an object a simple and practical diagnostic aidfor use in the detection of bacteria in biological and the other fluids.

Another object of this invention is a simple, practical and reliablemethod for the detection of bacteria in biological and other fluids.

A further object of the invention is a simple and practical method forproducing a diagnostic aid for use in the detection of bacteria inbiological and other fluids.

A still further object of the invention is a class of chemical compoundswhich are metabolized by bacteria to produce colored dyestufis, theintensity and nature of which can be used for quantitative and sometimesqualitative detection of bacteria.

These and other objects will be apparent from a consideration of thefollowing disclosure:

In accordance with the invention, it has now been found that compoundsof the formula:

wherein X is oxygen, or an alkylated nitrogen atom and R is one of thefollowing radicals:

OH= =OH NO,

gHs

wherein R is a saturated or unsaturated aliphatic, aromatic,

araliphatic, acyl, hydroxy or acyloxy-aliphatic radical, R, is ahydrogen atom or a lower alkyl radical, R and R are both hydrogen atomsor together form an isopropylidene radical and n is 2 or 3, are suitablefor use in the detection of bacteria in biological fluids such as spinalfluid, peritoneal fluid, serum and urine, and other fluids such as milk,water, infusion liquids and the like, in that they gave rise to a colorreaction in the presence of bacterial microorganisms.

This new means for the detection of bacteria depends upon the fact thatthe compounds of formula l are metabolized by bacteria to producedyestuffs, the reaction being, in some cases, even specific for strainsof bacteria.

The process according to the present invention for detecting bacteria iscarried out by bringing together the biological fluid to be investigatedwith a liquid or solid nutrient medium and a nongrowth-inhibiting amountof a compound of formula (1). Following an incubation time of -l8 hoursat 20-37 C., the color change is evaluated.

The diagnostic agents according to the present invention comprise aliquid or solid nutrient medium and a nongrowthinhibiting amount of acompound of formula l Using the new test procedure according to thepresent invention, there can be carried out a process for generallydetecting bacteria, i.e., a qualitative total determination ofmicro-organisms. in other words, it is possible to detect a plurality ofmicro-organisms which can, for example, be present at the same time inthe biological fluid as a result of an infection of the urinary tract,by means of a common color reaction. However, there can also be achieveda differentiation of the micro-organisms, i.e., a qualitative detectionof individual types of micro-organisms. in other words, individual typesof micro-organisms, for example, only Escherichia coli, can, on thebasis of a color reaction specific for the micro-organisms in question,be detected and exactly determined. The sample of biological fluid to betested can, in this connection, also be tested successively with severaldifferent diagnostic agents according to the present invention, thosecompounds of structural formula (1) being advantageously chosen whichgive easily difierentiateable, specific color reactions. in addition,preferred forms of these combinations also contain selectively effectivebactericidal or bactcriostatically effective substances and/ordifferentiating nutrient media which provide especially favorable growthconditions for the type of micro-organism to be detected but whichinhibit undesired accompanying microorganisms.

Since, in the case of certain of the compounds having structural formula(1), the time for the coloration to appear depends upon the number ofmicro-organisms originally present, the process according to the presentinvention carried out with the diagnostic agents herein disclosed,provides a means whereby semiquantitative determinations of the numberof micro-organisms is also possible.

In carrying out the process according to the present invention, thesolution to be analyzed, such as, for example, urine, milk, water or thelike, is added to a nutrient broth which is contained, for example, in atest tube, the broth having present therein a sufficient butnongrowth-inhibiting amount of one of the compounds of structuralformula (I). An upper limitation of the amount of compounds of formula(1) to be added is necessary because the compounds l possess certainbactericidal or bacteriostatic properties. Because of this fact, it was,indeed, to have been expected that, in the presence of these compounds,bacterial growth would no longer have been possible so that thecompounds also could not be metabolized by bacteria thereby giving riseto the colored dyestuffs. Surprisingly, however, for the detectionreaction according to the present invention, there are necessary amountswhich are substantially smaller than the minimum inhibitingconcentrations and no disturbances or blocks occur in carrying out thetest procedure.

Certain of the compounds of structural formula l) are only sparinglysoluble. in such instances, it is advantageous to add a solubiliier inorder to provide sufficient concentrations of the compounds for thecolor reaction. The test mixture is left for a few hours at roomtemperature or in an incubation oven, or bath at a temperature of 37 C.and the color changes evaluated from time to time. instead of a liquidnutrient broth, the compounds of structural formula l can also be mixedwith a warmed, liquid nutrient agar and, after cooling, the liquid to betested applied onto the solidified nutrient medium.

Instances of preferred forms of the diagnostic agents according to thepresent invention include nutrient-containing tablets ornutrient-containing filter paper strips which already contain a compoundhaving the structural formula (1). If a tablet is used for carrying outthe detection reaction in accordance with the invention, the same needonly be dissolved in a sample of the liquid to be tested. After anincubation time of from I -l8 hours, the color change can be evaluateddirectly. in the case of the use of filter paper test strips, the stripsare dipped for a short time in the solution to be tested, then placed ina small, sterilized test tube and kept for some time at room temperatureor at 37 C. The result can then be read off directly. In order tomaintain the tablets or filter paper strips storable,

ethylene oxide and sealed between sterilized foils which can easily beremoved prior to use.

The advantages of the process according to the present invention and ofthe diagnostic agents of the present invention are, in the first place,the simple and practical manner in which they can be handled, as well astheir reliability in indicating that micro-organisms are present, theamount and nature thereof. in particular, the causal micro-organismswhich occur in the case of nonspecific infections of the urinary tract,such as Escherichia coli, Streptococcus faecalr's, Proteus mirabilis,Staphylococcus aureus, Klebsiella pneumonia and Pseudomonas aemginosa,can be dependably detected. Furthermore, according to the presentinvention it is also possible not only to detect the general presence ofbacteria but also to carry out qualitative and semiquantitativeindividual determinations of special types of micro-organisms, withoutfirst having to carry out laborious, expensive and time-consumingisolations of the causal micro-organisms in pure cultures.

It is also possible to carry out the detection reactions, without theuse of an incubation oven or bath, at room temperature but in such casessomewhat longer incubation periods must be used. Whereas with the use ofan incubation oven, times of only 1 -l2 hours are necessary, at roomtemperature it is usually necessary to permit from 4 -l 8 hours toelapse in order that a clear coloration be obtained. In the case of theuse of prepared nutrient media, test papers and tablets, the usualpreparation times are rendered unnecessary and it is necessary only touse test tubes and/or pipettes in carrying out the determinations.

It has also been found that the incubation times required in actualpractice can be considerably shortened and a clearer distinction betweennormal and asymptomatic/significant ranges of the number ofmicro-organisms can be achieved when the material to be investigated,such as a nutrient broth containing micro-organisms or urine containingmicro-organisms which has been mixed with a nutrient medium, isincubated without the addition of one of the compounds having structuralformula (I), after which a compound of formula (1) is added, incubationcontinued for a short period of time and the color change thenevaluated.

in the case where a urine sample is being analyzed, a period of about 9l0 hours is necessary when a compound of structural formula (1) is addedimmediately, whereas with the use of this modified procedure, the endresult can be obtained after only about 5 6 hours.

Finally, it has also been found that the incubation time required inactual practice can also be significantly reduced and a clearerdistinction between normal and asymptomatic/significant" ranges of thenumber of micro-organisms can be achieved when the material to beinvestigated, such as a nutrient broth containing microorganisms orurine containing micro-organisms, which has been mixed with a nutrientmedium, is additionally mixed with coenzymes, as well as, if desired, asulthydryl group-containing compound and/or enzyme-activating heavymetal ions.

Coenzymes, which can be used in the foregoing procedure, include, forexample, nicotine-adenine-nucleotide (NAD) ornicotine-adenine-nucleotida phosphate (NADP), or the reduction productsthereof.

As sulfhydryl group-containing compounds, there are preferably usedcysteine, reduced glutathione, cysteamine or B-mercapto-ethanol. inplace of the compounds having free sulihydryl groups, there can also beused the corresponding dehydrogendated disulfide compounds.

Enzyme-activating heavy metal ions, which are suitable for use inaccordance with the invention, are preferably provided by the salts ofmanganese and copper.

These additives accelerate the coloration, not only in the case in whicha compound having the formula (1) is present initially, but also in thecase in which a compound of fonnula (l) is added after a preliminaryperiod of incubation. Since the time factor plays a decisive part forthe practical applicathey are advantageously sterilized with tion of theprocess according to the present invention, the

working method using a preliminary incubation and subsequent addition ofa compound of structural formula (1) is preferably further acceleratedand improved by the addition to the nutrient medium of theabove-mentioned further additives.

Excellent results are even obtained by the addition of the coenzymesalone. However, optimum results are obtained when, in addition, asulfhydryl group-containing compound and heavy metal ion, preferably inthe form of a manganous salt, are also present.

Thus, for example, after a preliminary incubation of only 1 -2 hours at37 C., the addition of a nongrowth-inhibiting amount of a compound ofstructural formula (1) and a subsequent incubation for 30 minutes at 37C. pen'nits the clear recognition of a large number of micro-organisms.1n the case of incubating for a further 1 -2 hours, smaller numbers ofmicroorganisms of up to 100,000 per milliliter, which are, however, alsoof interest in practice, can also be identified.

For this variant of the process according to the present invention, ithas proved to be particularly useful to employ gelatine capsules whichcontain all the components of the nutrient broth and the above-mentionedadditives. The contents of a capsule, which are sufficient for use inone determination, are shaken out into the fluid specimen. After thepreliminary incubation period, a compound of structural formula (1preferably in the form of a stable solution, is added thereto. After arelatively short further period of incubation, the coloration isevaluated.

The compounds of structural formula 1) are new. They can be preparedusing the known methods as, for example, described in the following:

1. General procedure for the preparation of N-substituted2,4-di-(5-nitro-2-furfurylidene)-granatan-3-one and 2,4-di-(5-nitro-2-furfurylidene)-tropinone derivatives of formula 1(a):

0.1 mol of an N-substituted norgranatan-3-one or tropinone (see infra)was heated under reflux and with stirring for 3 hours with 28.2 g. (0.2mol) 5-nitrofurfural in 50 ml. acetic anhydride. The reaction mixturewas thereupon stirred with ice water, the precipitated crystallinesubstance filtered off with suction and recrystallized. The compoundsset out in the following table were prepared in this manner:

was dissolved in 100 ml. acetic anhydride. Seven g. (0.05 mol)5-nitrofurfural were added thereto and the reaction mixture heated to 70C. After some time, the precipitated material was filtered off withsuction, washed with ether and recrystallized. The compounds set out inthe following table were prepared in this manner:

TABLE Period of Compound reaction, Yield, M.P.. Recrystal- No. 2 -hnurspercent C. lizedlrom II 5 80 278 Acetic acid. XIII CH3 1.5 41 226-227Dioxan.

Ill. l-phenyl-3-methyl-4-(5-nitro-2-fiirfurylidene)-pyrazol- S-one offormula l(c) (compound no. XIV):

17.4 g. (0.1 mol) l-phenyl-3-methyl-pyrazol-S-one and 14.1 g. (0.1 mol)5-nitrofurfural in 150 ml. acetic anhydride were stirred for 3 hours at100 C. After cooling, the reaction mixture was filtered with suction and28.7 g. of crude product were recovered. After recrystallization fromdioxan, there was obtained 1phenyl-3-methyl-4-(5-nitro-2-furfurylidene)- pyrazol-3one in a yield of55.1 percent of theory. This compound melted at 234 C., withdecomposition.

1V. 3-(5-nitro-2-thienyl)-2-nitro-propane-l,3-diol and 2,2-

dimethyl-4-(S-nitro-Z-thienyl)-5-nitro-1,3-dioxan of formula l(c)(compounds nos. XV and XVI):

6.28 g. 5-nitro-thiophane-2-aldehyde were dissolved in 40 ml.chloroform, 4.4 g. nitro-ethanol, dissolved in 20 ml. chloroform wereadded thereto, and the reaction mixture mixed with 0.4 -0.6 ml.triethylamine and allowed to stand at room temperature. After only ashort time, an oil precipitated out which, after cooling andtriturating, crystallized. After standing for several hours in arefrigerator, the product was filtered off with suction, washed withchloroform. There were thusly obtained 9.1 g. (91.8 percent of theory)of crude product having a melting 'point of l l l-l 14 C. Afierrecrystallization from isopropanol-water (1:1), with the addition of atrace of p-toluene-sulfonic acid, there was obtained analytically-pure3-(5-nitro-2-thienyl)-2-nitro-propanel ,3- diol having a melting pointof 136-140 C.

1.24 g. of the crude diol obtained as above (mp. 11 1l 14 C.) weredissolved in 6 ml. anhydrous acetone, 12 ml. an-

TABLE Compound Yield, No. R; X 11 percent M.P., C. Recrystallized from ICH2CH:OCOCH3 0 3 83 144-145 Alcohol/glacial acetic acid. 11.. -CH 0 3 63226-228 Isopropauol. I1I.. CH3 0 2 18 203-204 Isopropanol. IV C2Hs 0 356 226-227 Dlrnethylformamlde. V -CiH 0 3 5 218-220 Butanol. VI, CH2CoH50 3 74 215-217 Dioxan. VII -CH2CHCH2 0 3 37 186187 Iso ropanol. VIIL- OH0 3 9 1 254 To uene. IX CH2CH2C0H5 0 3 94 158-159 Isopropanol. X C CH3 03 14 255 Acetic acid. XI -CHa S 3 43.3 210-212 Acetic acid.

material for the preparation of compound (VIII) is also new and can beprepared in the following manner:

146 g. (1 mol) acetone dicarboxylic acid, 56 g. sodium acetatetrihydrate, 69.6 g. hydroxylamine hydrochloride and 400 ml. 25 percentglutaric dialdehyde solution were dissolved in 1,000 ml. water. Theevolution of carbon dioxide set in immediately. The solution was allowedto stand for 3 days at room temperature 1t was then rendered alkalinewith a solution of sodium hydroxide and extracted with chloroform. Theextract was dried and evaporated. As residue, there were obtained 108.6g. N-hydroxy-norpseudopelletierine. The compound is recrystallized fromethyl acetate and had a melting point of l37-139 C. The yield ofN-hydroxy-norpseudopelletierine amounted to 5 1 percent of theory.

11. General procedure for the preparation of 5-nitrofurfuryli-denederivatives of homophthalimide of formula l(b):

0.05 mol homophthalimide or N-methyl-homophthalimide TheN-hydroxy-no$seudopelletierine required as starting hydrous benzene, andan intimate mixture of 0.6 g. phosphoric pentoxide and 0.6 g.kieselguhr, were added thereto. The reaction mixture was heated to 4045C. After 15 minutes, a further 12 ml. benzene was added and the reactionmixture thereafter stirred for about 6 hours at 4045 C. Any undissolvedmaterial was then filtered off with suction and washed with ethylenechloride into the filtrate. The filtrate was neutralized with an aqueousslurry of calcium carbonate and the undissolved material presentfiltered off with suction. The aqueous phase of the filtrate wasextracted with ethylene chloride. The organic phases were combined,dried over anhydrous sodium sulfate and evaporated in a vacuum, 1.4 g.of an oil thereby remaining behind. This was first triturated withligroin and then with isopropyl ether, the crystals thus obtainedfiltered off with suction and then washed with isopropyl ether. Therewere thus obtained 0.8 g. of crystals (55.5 percent of theory) having amelting point of l05-l08 C. Following recrystallization fromisopropanol, the melting point of the 2,2-dimethyl-4(5-nitro-2-thienyl)-5-nitro- 1 ,3-dioxan obtamed increased to l07-l09 C. (decomp.). Theyield amounted 100.55 3.

V. l-( S-nitrol -methyl-2-pyrryl )-2-( 4-quino1yl )-ethylene of formula1(d) (compound No. XVII):

1.68 g. 4-methyl-quinoline were heated for 4 hours under reflux (160 C.;bath temperature) with 1.85 g. 5-nitro-1- methyl-pyrrole-Z-aldehyde in12 ml. acetic anhydride. After cooling the resulting reaction mixture,the precipitated yellow crystals were filtered off with suction, washedwith a little acetic anhydride and then with isopropyl ether. There werethusly obtained 0.6 g. (percent of theory) of paper chromatographicallypure material. Following recrystallization from 17 ml. dioxan, with theaddition of activated charcoal, the compound melted at 240-242 C.(decomp.). The yield was 0.45 g.

The following examples are given for the purpose of illustrating theprocess according to the present invention and also the diagnosticagents according to the present invention. The examples are in nowise tobe construed in limitation of the invention:

EXAMPLE 1 General procedure for carrying out the detection of bacteria25.6 g. of a compound corresponding to structural formula (1) weredissolved in 5 ml. dimethyl formamide and 5 ml. polyoxyethylene sorbitanlaureate (Tween (a Registered Trademark of Atlas Powder Co.). Usingsterilized distilled water there was prepared a geometric dilutionseries (factor 0.5) which was diluted with a conventional nutrientbroth, such as that supplied by Difco, in a ratio of 1:10. Accordingly,the individual solutions contained 256, I28, 64, 32, 16, etc., ugJml. ofthe compound of formula (1 Sterilized test tubes were then each filledwith 2 ml. of these solutions and mixed with the bacteria-containingsample to be tested. The test tubes were sealed with a sterilizedcottonwool swab and incubated at 2l-37 C. In place of an incubationcabinet, there could also have been used a thermostatically controlledwater bath. in dependence upon the type of micro-organism, the period ofincubation and the number of micro-organisms initially introduced, thetest sample began to become colored. The color and the time ofcoloration were compared with standard values and, in this manner, thenature and number of the bacteria were indicated. Because of thebacteriostatic action of some of the compounds of formula (l) to certainstrains of micro-organisms, only rather weak colorations or nocolorations at all were produced in the test samples with highconcentrations of the compound of formula (2) being evaluated. Ingeneral, therefore, only the test samples with the lower concentrationswere evaluated.

In the following tables 1, II and 111, there are summarized the typicalcolorations depending upon the compound of formula TABLE I Streptococcusfaccali's Ecchanchia (cli (18) (155) (1) used, the nature of themicro-organism. the temperature and the incubation period. ln general,the number of micro-organisms is above 100,000/ml. i.e., themicro-organism number present in cases of significant bacterurias.Micro-organism numbers of less than 100,000/ml. down to 10.000/ml. arefound in cases of asymptomatic bacterurias and micro-organism numbersbelow 10,000/ml. are regarded as normal.

TABLE II Protozzs mirabilis Staphylococus aurcus Color- Color-Incubation ation ation begins Color begins Color Comtemp after alterpound (hrs) (hrs.)

I 37 4 12 Yellow-green.

21 12 12 DO. H 37 4 12 Green. 21 12 H... 0.-.-. 12 D0. III 37 4Red-brown 12 Brown-red. IV 37 4 Brown 12 Yellow-green. V.-. 37 18 VI 375 Violet. VII.-. 37 4 Yellow. VIIL. 37 5 IX 87 4 Brown.

21 12 Yellow-brown. X 37 18 XII. 37 6 Yellow-green. XIII 37 3 Red Olivegreen.

21 10 Red-brown 10 Greenish. XIV 37 18 Violet 18 Violet.

12 Orange. 7 Green. 12

TABLE III Klebsiella pncumoniae Pscudomona acruginosa Color- Color-Incuation at on batlon begins begins Comtemp. after afterpound in C.(hrs.) Color (hrs.) Color I 37 6 Violet-brown... 12 Greenish. 0. IIRed-brown.

Green-brown. III Brown. IV Grennlshbrown.

Red-brown. Brown. Yellow. Brown.

21 12 Yellow-brown.. 12 Yellow-orange. 37 18 Brown 37 7 Orange-red... 12Yellow-green. 37 3 Brown-green... 3 Green. 21 10 Yellow-green... 10 Do.37 18 Violet 18 Violet. 37 37 Yellow-green. 21

EXAMPLEZ Semiquantitative determination of the number of micro-organismsa. Escherichia coli (69) Compound XVll was used at a concentration of32;.tg./ml. and an incubating temperature of 37 C. When the number ofmicro-organisms amounted to 1,000 to 10,000/ml. no color change tookplace, even after 10 hours. In the case of microorganisms present in anamount of 10,000 to 50,000/ml. the yellowish solution took on a cleargreen color after 10 hours. When the number of micro-organisms presentwas 50,000 to 100,000/ml. this coloration occurred after only 9 hours,when the number of micro-organisms present was 100,000 to 500,000/ml.,coloration appeared after 8 hours, and when the number ofmicro-organisms was more than 500,000/ml. after only 7 hours.

b. Klebsiella pneumoniae (286) There was used compound 11 at aconcentration of 64 p.g./ml. and an incubation temperature of 37 C. Whenthe number of micro-organisms present was from 1,000 to 100,000/ml.after 9 hours. the yellowish solution became redhrown and after 10hours, violet. In the case where the microorganisms were present in anamount of 100,000 to more than 500,0()/ml., the red-brown color appearedafter only 7-8 hours, and the violet color after only 9 hours.

0. Proteus mirabilis (298) Compound XIII was employed at a concentrationof 32 pig/ml. and an incubation temperature of 37 C. In the case ofmicro-organisms present in an amount of 4,000,000/ml., the pale yellowsolution became red after only 4 hours. When the number ofmicroorganisms equalled 400,000ml., the color change occurred after 6hours, with 40,000/ml. the color change was apparent after 7 hours, with4,000/ml. after 8 hours, and with 400/ml. after 9 hours.

EXAMPLE 3 1.8 ml. of nutrient broth containing microorganisms wereincubated for hours at 37 C. Thereafter, 0.2 ml. of a solution whichcontained compound II in a concentration of 500 ug/ml. was added. (Forthe preparation of this solution, 10 mg. of compound II were dissolvedin 2 ml. dimethyl formamide and 2 ml. polyoxyethylene sorbitan laureate(Tween thereafter mixed with 6 ml. distilled water and then againdiluted with distilled water in a ratio of 1:1). After incubating forhalf an hour, the coloration was evaluated. In the following table,there are summarized the results of experiments with variousmicro-organisms and different numbers of micro-organisms.

TAB LE IV Number of mlcroorganisms/ml.'

50,000- 100,000 More than Test organism 100,000 500,000 500,000

Escherichia coli (18) Weak Violet.

Strcptococcus faecalis (155).. Proteus mirabilis (208) Staphylococcusaureus SG 511 Klcbsiciln pneumoniae (188) Violet.

As can be seen from this table, substantially clearer color changes areobserved in case micro-organisms are present in amounts which are ofsignificance in practice.

EXAMPLE 4 Using a procedure analogous to that described in example 3, anutrient broth containing micro-organisms or a urine sample containingmicro-organisms was incubated for 5 hours at 37 C., then mixed with 0.2ml. of a solution of compound XVII, incubated for a further half an hourand thereafter eval- I uated for color development. As in example 3, thesolution of compound XVII was prepared from 10 mg. of the compound IXVII in 2 ml. dimethyl formamide, 2 ml. polyoxyethylene sorbitanlaureate and 6 ml. distilled water followed by dilution with distilledwater in a ratio of 1:1. The results obtained with various types ofmicroorganisms and various numbers of micro-organisms are summarized inthe following table V:

As can be seen from this table, sharp limits of the color change areobserved in the instances where the numbers of micro-organisms presentare those of significance in practice.

EXAMPLE 5 In 1.8 ml. of a urine sample containing micro-organisms, therewere dissolved the contents of a gelatin capsule which consists of 10mg. soya peptone, 5 mg. dextrose, 10 mg. sodium chloride and 5 mg.dipotassium hydrogen phosphate, and additionally the following additiveswhich serve to promote the reaction: 4 mg. DPN, 1 mg. Cysteine and 0.1mg. manganous chloride tetrahydrate.

The sample was incubated for two hours at 37 C. and then mixed with 0.2ml. of a solution of compound II at a concentration of 500 ,u.g./ml.(For the preparation of this solution, 10 mg. of compound II weredissolved in 2 ml. dimethyl formamide and 2 ml. polyoxyethylene-sorbitanlaureate (Tween 20"). the solution was then mixed with 6 ml. distilledwater and thereafier again diluted with distilled water in a ratio of1:1). After an incubation time of half an hour, the coloration wasevaluated for the first time. Further evaluations were carried out afterfurther incubation for I and 2 hours.

In the following table VI, there are summarized the results of thisprocedure where the number of micro-organisms was about 3,000,000/ml.:

TABLE VI Color after turftlier incubation o A series of parallelexperiments using the same nutrient medium but without the additiveswhich act to promote the reaction was carried out. After such shortpreliminary incubation times, color changes which are visible to thenaked eye could not be ascertained.

EXAMPLE 6 The color which is formed when microorganism-containingsolutions were incubated with compound II, could be measured in aphotometer at 578 nm. even in the case of quite low concentrations. Thefollowing measurements, which were carried out on an aqueous suspensioncontaining 30 million micro-organisms per milliliter with the immediateaddition of compound II, establish that, by the addition of I mg. DPN orTPN, the development of the color commences substantially earlier. It isto be noted that at an extinction of about E=0.200, a change from yellowto violet can be clearly observed by the naked eye. The results obtainedare set out in the following table VII:

In the same manner as described in example 6, bacterial suspensions ofEscherichia coli (18) which contain 630,000 micro-organisms permilliliter and increasing amounts of DPN were incubated and evaluatedphotometrically. The results which were obtained are set out in tableVIII.

EXAMPLE 8 Urine samples from clinical patients in which micro-organismnumbers of between 10,000 and 3,000,000 per milliliter had beendetermined in the conventional manner, without a specification of thenature of the micro-organisms having been established, were investigatedin the manner described in example 5. After a preliminary incubation ofonly 2 hours and a further incubation of half an hour, the urine samplesshowed a violet color, indicating more than 2,000,000micro-organisms permilliliter. Urine samples which contained about 100,000 micro-organismsper milliliter became violet colored after a further incubation periodof 1-2 hours.

All of the compounds of formula (I) are new and can be used as describedherein for the detection of bacteria in biological and other fluids.

However, compounds corresponding to formula la) i.e.,

wherein X is an oxygen, sulfur or an alkylated nitrogen atom and R is analiphatic, aromatic, araliphatic, acyl, hydroxy, or acyloxy substitutedaliphatic radical and n is 2 or 3 are particularly effective whenemployed as herein described for the detection of bacteria.

Preferred solubilizers for the new compounds are besides polyoxyethylenesorbitan laureate (Tween 20) condensation products of higher alcoholswith ethyleneoxide (Emulgin) (a Registered Trademark of Dehydag,Dusseldorf) and mixtures of dioxane with polycthyleneglycole 6000.

Preferred liquid nutrient broths, as used within the scope of thisinvention, are for example that of Difco, containing within 1 liter g.peptone, 5 g. sodium chloride, 2g. dextrose, 2.5 g.

dipotassium hydrogen phosphate and an extract of calves brains and neatshearts, or Nutrient Broth C M l,Oxoid, containing 5 g. peptone, l g.extract of meat, 5 g. sodium chloride and 2 g. extract of yeast. Atypical example of a solidified nutrient medium contains 15.6 g.peptone, 5.6 g. sodium chloride, 1 g. dextrose, 2.8 g. extract of yeastand 30 g. agaragar.

We claim:

1. A compound of the formula wherein X is a member selected from thegroup consisting of oxygen, sulfur and methylated nitrogen atoms andwherein R, is a member selected from the group consisting of loweralkyl, lower alkenyl, phenyl, phenyl-lower alkyl, lower alkanoyl,hydroxy and lower alkanoyloxy-lower alkyl radicals.

2. A compound according to claim 1 designated N-( 2-acetoxyethyl)-2,4-bis-(S-nitro-Z-furfurylidene)-norgranatan- 3-one.

3. A compound according to claim 1 designated 2,4-bis-(5-nitro-2-furfurylidene)-granatan-3-one.

4. A compound according to claim 1 designated 2,4-bis-(5-nitro-Z-furfurylidene)-tropin-3-one.

5. A compound according to claim 1 designated N-ethyl-2,4-bis-(5-nitro-2-furfurylidene)-norgranatan-3-one.

6. A compound according to claim 1 designated N-phenyl-2,4-bis-(S-nitro-Z-furfurylidene)-norgranatan-3-one.

7. A compound according to claim 1 designated N-benzyl-2,4-bis-(5-nitro-2-furfurylidene)-norgranatan-3-one.

8. A compound according to claim 1 designated N-allyl-2,4-bis-(5-nitro-2-furfurylidene)-norgranatan-3-one.

9. A compound according to claim 1 designated N-hydroxy-2,4-bis-(5-nitro-2-furfurylidene)-norgranatan-3-one.

10. A compound according to claim 1 designated N-(B- phenethyl)-2,4-bis-( 5-nitro-2-furfurylidene )-norgranatan-3- one.

11. A compound according to claim 1 designated N-acetyl- 2,4-bis-(5-nitro-2-furfurylidene )-norgranatan-3-one.

12. A compound according to claim 1 designated, 2,4-bis-(5-nitro-2-thienylidene)-granatan-3-one.

I Boehr. 280.1

PMOW --UNITED STATES PNiEN-T @FFECE 9 CERTIFICATE OF CORRECTION ParentNo. 3,62%016 be ted November 16, 19 7 1 lnvemofls) Herbert Berger, OttoBold, Dietrich Kruger, Kurt S tach,

Felix Helmut Schmifit, Harald Stork it is certified that error appearsin the above-identified patent and that said Letters Patent are herebycorrected as shown below:

Abstract, first line after formula (Spee. page 2, line 1) "where" shouldbe --wherein-- Col. 6 line 22 (Spec. p. 17, 115% 9/10 "pyrezol-Sone"should be --pyrazo1-5-one- Riel 7' line 11 (Spec. p. 19, line a (10percent: of theory) shoulfi be "(187. of theory) Signed and sealed this23rd day of May 1972.

. icteet:

EDJIARD BLFLETCEER ,JR. ROBERT GOTTSGHALK Actestlng Off1@@r Commissionerof Patents

2. A compound according to claim 1 designatedN-(2-acetoxyethyl)-2,4-bis-(5-nitro-2-furfurylidene)-norgranatan-3-one.3. A compound according to claim 1 designated2,4-bis-(5-nitro-2-furfurylidene)-granatan-3-one.
 4. A compoundaccording to claim 1 designated2,4-bis-(5-nitro-2-furfurylidene)-tropin-3-one.
 5. A compound accordingto claim 1 designatedN-ethyl-2,4-bis-(5-nitro-2-furfurylidene)-norgranatan-3-one.
 6. Acompound according to claim 1 designatedN-phenyl-2,4-bis-(5-nitro-2-furfurylidene)-norgranatan-3-one.
 7. Acompound according to claim 1 designatedN-benzyl-2,4-bis-(5-nitro-2-furfurylidene)-norgranatan-3-one.
 8. Acompound according to claim 1 designatedN-allyl-2,4-bis-(5-nitro-2-furfurylidene)-norgranatan-3-one.
 9. Acompound according to claim 1 designatedN-hydroxy-2,4-bis-(5-nitro-2-furfurylidene)-norgranatan-3-one.
 10. Acompound according to claim 1 designated N-( Beta-phenethyl)-2,4-bis-(5-nitro-2-furfurylidene)-norgranatan-3-one.
 11. Acompound according to claim 1 designatedN-acetyl-2,4-bis-(5-nitro-2-furfurylidene)-norgranatan-3-one.
 12. Acompound according to claim 1 designated,2,4-bis-(5-nitro-2-thienylidene)-granatan-3-one.